Extracellular Enzymatic Hydrolysis Research Articles

Effect of Curcumin on Cognitive Behavior and Pathological Characteristics of the Hippocampus in Mice with Inherent Alzheimer's Disease.

This study examined the effect of curcumin on pathological manifestations and clearance of amyloid β peptide (Aβ) in the hippocampus of 8-month-old transgenic APP/PS1 mice with inherent Alzheimer's disease. APP/PS1 mice and the age-matched wild-type controls were subjected to 3 behavioral tests: open field, new object recognition, and Morris water maze. Expression of Aβ, APP, CTF, BACE1, IDE, NEP, and LRP1 proteins in the extracted hippocampal tissue was evaluated by Western blotting. The distribution and the quantity of amyloid plaques and the spread of microglia in the hippocampus were determined by immunofluorescence. The contents of Aβ40 and Aβ42 in the hippocampus were assayed and analyzed on Simoa HD-1 analyzer. The proteins interacting with Aβ in the hippocampus of APP/PS1 mice were detected by co-immunoprecipitation. Curcumin significantly reduced motor hyperactivity in the open-field test, improved short-term recognition memory, spatial learning, and reference memory in APP/PS1 mice. In the hippocampus of APP/PS1 mice, curcumin significantly diminished the elevated Aβ levels and inhibited microglia proliferation. At the same time, curcumin had no effect on Aβ production, extracellular enzymatic hydrolysis, and LRP1-mediated outward transport, but enhanced Aβ clearance by activation of the intracellular ubiquitin-proteasome system and related peripheral mechanisms. Thus, curcumin improves the learning and memory abilities of APP/PS1 mice and reduces the pathological accumulation of Aβ in the brain.

Bioinspired nano-vaccine construction by antigen pre-degradation for boosting cancer personalized immunotherapy

Cancer vaccines-based cancer immunotherapy has drawn widespread concern. However, insufficient cancer antigens and inefficient antigen presentation lead to low immune response rate, which greatly restrict the practical application of cancer vaccines. Here, inspired by intracellular proteasome-mediated protein degradation pathway, we report an antigen presentation simplification strategy by extracellular degradation of antigen proteins into peptides with proteolytic enzyme for improving the utilization of cancer antigens and arousing restricted cancer immunity. The pre-degraded antigen peptides are first validated to exhibit an increased capacity on antigen-presenting cell (APC) stimulation compared with proteins and still reserve antigen specificity and major histocompatibility complex (MHC) affinity. Furthermore, by coordinating the pre-degraded peptides with calcium phosphate nanoparticles (CaP), a CaP-peptide vaccine (CaP-Pep) is constructed, which is verified to induce an efficient personalized immune response in vivo for multi-model anti-cancer therapy. Notably, this bioinspired strategy based on extracellular enzymatic hydrolysis for vaccine construction is not only applicable for multiple types of cancers, but also shows great potential in expanding immunology fields and translational medicine.

Relative Importance of Phosphodiesterase vs. Phosphomonoesterase (Alkaline Phosphatase) Activities for Dissolved Organic Phosphorus Hydrolysis in Epi- and Mesopelagic Waters

Marine microbes use extracellular phosphatases to hydrolyse phosphate from organic matter. Dissolved organic phosphorus (DOP) is typically present in higher concentrations than phosphate in oceanic surface waters. Yet, the fate and role of different DOP components, such as phosphomonoester and phosphodiester, are poorly understood. Most of the investigations on extracellular enzymatic hydrolysis of marine DOP have focused on phosphomonoesterase (MEA) activity (i.e., alkaline phosphatase), whereas phosphodiesterase (DEA) measurements are scarce. This limits our understanding of the ecological and biogeochemical role of DOP sources other than P-monoesters in the sea. We determined extracellular MEA and DEA activities including their cell-free fractions on a bimonthly basis over 14 months in surface and mesopelagic subantarctic waters, thus covering a wide range of phosphate availability levels (from <0.5 to 2.3 µM). We found that DEA and MEA exhibit similar hydrolysis rates in surface as well as in mesopelagic waters. The MEA : DEA ratio varied between 0.38 and 5.42 during the study period, indicating potential differences in function and/or expression among the two enzyme groups, potentially reflecting differences in the availability and/or utilization of P-monoester and P-diester pools. Interestingly, the MEA : DEA was negatively correlated to phosphate (r=-0.82, p=0.02, R2=0.67) and positively with N:P (r=0.84, p=0.02, R2=0.67), suggesting that the relative importance of DEA versus MEA is linked to inorganic P availability and the N : P ratio. DEA was also related to the N : P ratio, both at the surface and at depth, suggesting DEA alone is sensitive to changes in the N : P ratio. The majority (>70%) of extracellular MEA and DEA was found in the cell-free fraction which increased with depth. Our results indicated that DOP hydrolysis mediated by DEA in the surface as well as in dark ocean is as important as the frequently measured MEA.

Molecular Basis for Substrate Recognition and Catalysis by a Marine Bacterial Laminarinase.

Laminarin is an abundant algal polysaccharide that serves as carbon storage and fuel to meet the nutrition demands of heterotrophic microbes. Laminarin depolymerization catalyzed by microbial extracellular enzymes initiates remineralization, a key process in ocean biogeochemical cycles. Here, we described a glycoside hydrolase 16 (GH16) family laminarinase from a marine alga-associated Flavobacterium at the biochemical and structural levels. We found that the endolytic enzyme cleaved laminarin with a preference for β-1,3-glycoside linkages and showed transglycosylation activity across a broad range of acceptors. We also solved and compared high-resolution crystal structures of laminarinase in the apo form and in complex with β-1,3-tetrasaccharides, revealing an expanded catalytic cleft formed following substrate binding. Moreover, structure and mutagenesis studies identified multiple specific contacts between the enzyme and glucosyl residues essential for the substrate specificity for β-1,3-glucan. These results provide novel insights into the structural requirements for substrate binding and catalysis of GH16 family laminarinase, enriching our understanding of bacterial utilization of algal laminarin.IMPORTANCE Heterotrophic bacterial communities are key players in marine biogeochemical cycling due to their ability to remineralize organic carbon. Processing of complex organic matter requires heterotrophic bacteria to produce extracellular enzymes with precise specificity to depolymerize substrates to sizes sufficiently small for uptake. Thus, extracellular enzymatic hydrolysis initiates microbe-driven heterotrophic carbon cycling. In this study, based on biochemical and structural analyses, we revealed the depolymerization mechanism of β-1,3-glucan, a carbon reserve in algae, by laminarinase from an alga-associated marine Flavobacterium The findings provide new insights into the substrate recognition and catalysis of bacterial laminarinase and promote a better understanding of how extracellular enzymes are involved in organic matter cycling.

Differential visible spectral influence on carbon metabolism in heterotrophic marine flavobacteria.

The visible spectrum of solar radiation is known to stimulate photoheterotrophic bacterial carbon metabolism. However, its impact on 'strictly' heterotrophic bacteria remains less explored. Here, we show that heterotrophic flavobacteria exhibit enhanced uptake and mineralization of dissolved organic carbon with increasing wavelengths of visible light, without employing any 'known' light-harvesting mechanisms. RNA sequencing identified blue light as a major constraint in the extracellular enzymatic hydrolysis of polymeric carbohydrates and acquisition of sugars, despite acting as a stimulus for inorganic carbon sequestration. In contrast, green-red and continuous full-spectrum lights activated diverse hydrolytic enzymes and sugar transporters, but obstructed inorganic carbon fixation. This 'metabolic switching' was apparent through limited nutrient uptake, suppressed light-sensitivity, oxidative stress response and promotion of inorganic carbon sequestration pathways under blue light. The visible light impact on metabolism may be of significant ecological relevance as it appears to promote cell-mediated mineralization of organic carbon in 'green-colored' chlorophyll-rich copiotrophic coastal seawater and inorganic carbon sequestration in 'blue-colored' oligotrophic open ocean. Thus, a novel regulatory role played by light on heterotrophic metabolism and a hidden potential of flavobacteria to sense and respond differentially to monochromatic lights influencing marine carbon cycling were unraveled.

Hydrolysis of Ionic Phthalic Acid Based Polyesters by Wastewater Microorganisms and Their Enzymes.

Water-soluble polyesters are used in a range of applications today and enter wastewater treatment plants after product utilization. However, little is known about extracellular enzymes and aquatic microorganisms involved in polyester biodegradation and mineralization. In this study, structurally different ionic phthalic acid based polyesters (the number-average molecular weights (Mn) 1770 to 10 000 g/mol and semi crystalline with crystallinity below 1%) were synthesized in various combinations. Typical wastewater microorganisms like Pseudomonas sp. were chosen for in-silico screening toward polyester hydrolyzing enzymes. Based on the in-silico search, a cutinase from Pseudomonas pseudoalcaligenes (PpCutA) and a putative lipase from Pseudomonas pelagia (PpelaLip) were identified. The enzymes PpCutA and PpelaLip were demonstrated to hydrolyze all structurally different polyesters. Activities on all the polyesters were also confirmed with the strains P. pseudoalcaligenes and P. pelagia. Parameters identified to enhance hydrolysis included increased water solubility and polyester hydrophilicity as well as shorter diol chain lengths. For example, polyesters containing 1,2-ethanediol were hydrolyzed faster than polyesters containing 1,8-octanediol. Interestingly, the same trend was observed in biodegradation experiments. This information is important to gain a better mechanistic understanding of biodegradation processes of polyesters in WWTPs where the extracellular enzymatic hydrolysis seems to be the limiting step.

Combined Carbohydrates Support Rich Communities of Particle-Associated Marine Bacterioplankton.

Carbohydrates represent an important fraction of labile and semi-labile marine organic matter that is mainly comprised of exopolymeric substances derived from phytoplankton exudation and decay. This study investigates the composition of total combined carbohydrates (tCCHO; >1 kDa) and the community development of free-living (0.2–3 μm) and particle-associated (PA) (3–10 μm) bacterioplankton during a spring phytoplankton bloom in the southern North Sea. Furthermore, rates were determined for the extracellular enzymatic hydrolysis that catalyzes the initial step in bacterial organic matter remineralization. Concentrations of tCCHO greatly increased during bloom development, while the composition showed only minor changes over time. The combined concentration of glucose, galactose, fucose, rhamnose, galactosamine, glucosamine, and glucuronic acid in tCCHO was a significant factor shaping the community composition of the PA bacteria. The richness of PA bacteria greatly increased in the post-bloom phase. At the same time, the increase in extracellular β-glucosidase activity was sufficient to explain the observed decrease in tCCHO, indicating the efficient utilization of carbohydrates by the bacterioplankton community during the post-bloom phase. Our results suggest that carbohydrate concentration and composition are important factors in the multifactorial environmental control of bacterioplankton succession and the enzymatic hydrolysis of organic matter during phytoplankton blooms.

Effects of elevated CO2 concentrations on the production and biodegradability of organic matter: An in-situ mesocosm experiment

The increase in atmospheric CO2 concentration is responsible for progressive ocean acidification and has potential widespread effects on organic matter production and degradation. In order to understand the effect of elevated CO2 concentrations on organic matter dynamics, we conducted an in-situ mesocosm study in the mid-latitude coastal seas. Changes in the concentration and characteristics of organic matter, including particulate and dissolved fractions, as well as extracellular enzymatic hydrolysis of polysaccharides, were determined at six pCO2 levels ranging from 260 to 1040ppmv over 19days. The phytoplankton bloom and accumulation of dissolved organic carbon were observed in all pCO2 treatments. In the present study, although the elevated pCO2 positively influenced the quantity of organic matter, it did not affect its fluorescence characteristics and biodegradability. The highest hydrolysis potentials for polysaccharide degradation were recorded under highly acidified conditions (890 and 1040ppmv). Our findings suggest that elevated CO2 concentrations could stimulate organic matter production and degradation, which in turn could lead to increased rates of organic carbon cycling.

Comparing extracellular enzymatic hydrolysis between plain peptides and their corresponding analogs in the northern Gulf of Mexico Mississippi River plume

Peptide analogs, such as leucine-methylcoumarinylamide (Leu-MCA) and Lucifer yellow anhydride-tetraalanine (LYA-Ala4), are often used as proxies to quantify extracellular peptidase activities, but how accurately these analogs represent natural peptides remains unclear. Here we compared hydrolysis rates of tetrapeptides alanine-valine-phenylalanine-alanine (AVFA) and tetraalanine (Ala4) with their corresponding LYA analogs along a salinity gradient in the northern Gulf of Mexico Mississippi River plume. Hydrolysis rates were generally similar, but not always, between natural peptides and their analogs in the plume water. For example, hydrolysis rates were similar between LYA-AVFA and AVFA at all stations except one. In contrast, hydrolysis rates of LYA-Ala4 were significantly higher than those of Ala4 at low-salinity (18–19) stations, but significantly lower at one high-salinity (36) station. These results suggest that relative activities of different types of peptidases, measured by the analogs or peptides, depended on biological and chemical properties at each station, such as abundance of certain “opportuni-trophic” bacteria, including Flavobacterium, Ruegeria and Roseobacter. The produced fragments and amino acids from peptide hydrolysis implied further that the hydrolysis pathway depended on specific peptide substrate, and that the LYA tag affected the hydrolysis pathway. Taken together, this study validates the technique of using LYA analogs to study extracellular enzymatic activities, particularly the overall hydrolysis rate, and provides insights into the patterns of extracellular enzymatic activities in estuarine environments.

Patterns of Microbially Driven Carbon Cycling in the Ocean: Links between Extracellular Enzymes and Microbial Communities

Heterotrophic microbial communities play a central role in the marine carbon cycle. They are active in nearly all known environments, from the surface to the deep ocean, in the sediments, and from the equator to the Poles. In order to process complex organic matter, these communities produce extracellular enzymes of the correct structural specificity to hydrolyze substrates to sizes sufficiently small for uptake. Extracellular enzymatic hydrolysis thus initiates heterotrophic carbon cycling. Our knowledge of the enzymatic capabilities of microbial communities in the ocean is still underdeveloped. Recent studies, however, suggest that there may be large-scale patterns of enzymatic function in the ocean, patterns of community function that may be connected to emerging patterns of microbial community composition. Here I review some of these large-scale contrasts in microbial enzyme activities, between high-latitude and temperate surface ocean waters, contrasts between inshore and offshore waters, changes with depth gradients in the ocean, and contrasts between the water column and underlying sediments. These contrasting patterns are set in the context of recent studies of microbial communities and patterns of microbial biogeography. Focusing on microbial community function as well as composition and potential should yield clearer understanding of the factors driving carbon cycling in the ocean.

Patterns of extracellular enzyme activities and microbial metabolism in an Arctic fjord of Svalbard and in the northern Gulf of Mexico: contrasts in carbon processing by pelagic microbial communities

The microbial community composition of polar and temperate ocean waters differs substantially, but the potential functional consequences of these differences are largely unexplored. We measured bacterial production, glucose metabolism, and the abilities of microbial communities to hydrolyze a range of polysaccharides in an Arctic fjord of Svalbard (Smeerenburg Fjord), and thus to initiate remineralization of high-molecular weight organic matter. We compared these data with similar measurements previously carried out in the northern Gulf of Mexico in order to investigate whether differences in the spectrum of enzyme activities measurable in Arctic and temperate environments are reflected in “downstream” aspects of microbial metabolism (metabolism of monomers and biomass production). Only four of six polysaccharide substrates were hydrolyzed in Smeerenburg Fjord; all were hydrolyzed in the upper water column of the Gulf. These patterns are consistent on an interannual basis. Bacterial protein production was comparable at both locations, but the pathways of glucose utilization differed. Glucose incorporation rate constants were comparatively higher in Svalbard, but glucose respiration rate constants were higher in surface waters of the Gulf. As a result, at the time of sampling ca. 75% of the glucose was incorporated into biomass in Svalbard, but in the northern Gulf of Mexico most of the glucose was respired to CO2. A limited range of enzyme activities is therefore not a sign of a dormant community or one unable to further process substrates resulting from extracellular enzymatic hydrolysis. The ultimate fate of carbohydrates in marine waters, however, is strongly dependent upon the specific capabilities of heterotrophic microbial communities in these disparate environments.

Degradation capability of the coastal environment adjacent to the Itata River in central Chile (36.5° S)

Abstract. The response of the coastal ocean influenced by both river discharges and inputs of photosynthetically derived organic carbon product of upwelling, was evaluated by estimating rates of microbial hydrolysis of macromolecules with the goal of estimating the potential degradation capability of the coastal ecosystem off central Chile. Extracellular enzymatic activity (EEA) in seawater was dominated by aminopeptidase activity on substrate L-leucine-4-methyl-7-coumarinylamide (MCA-leu) (1.2 to 182 nmol l−1 h−1) followed by 4-methylumbelliferyl-ß-D-glucoside (MUF-glu) (0.08–61 nmol l−1 h−1) and 4-methylumbelliferyl-ß-D-cellobiose (MUF-cel) (0.15–7 nmol l−1 h−1), with the highest rates measured during spring-summer. In riverine waters, extracellular enzymatic hydrolysis remained within the range of 45 to 131 nmol l−1 h−1 for MCA-leu and ca. 20 nmol l−1 h−1 for glucosidic substrates, year-round. Contrary to the EEA observed for the marine water column, surface sediment extracellular enzymatic hydrolysis of MCA-leu (0.04 to 6.13 nmol g−1 dw h−1) was in the same order of magnitude as the rates observed for MUF-cel (0.004 to 5.1 nmol g−1 dw h−1) and MUF-glu (0.007 to 10.5 nmol g−1 dw h−1). Moreover, hydrolysis in sediments was characterized by higher rates during winter compared with spring-summer in the coastal and estuarine zone. The five years of data allowed us to evaluate the potential capability of microbial processing of organic carbon in the coastal area adjacent to the Itata river discharge where the increase in primary production in the productive seasons is accompanied by the increase in hydrolysis of macromolecules.

Microbial Community Composition and Function in Permanently Cold Seawater and Sediments from an Arctic Fjord of Svalbard

Heterotrophic microbial communities in seawater and sediments metabolize much of the organic carbon produced in the ocean. Although carbon cycling and preservation depend critically on the capabilities of these microbial communities, their compositions and capabilities have seldom been examined simultaneously at the same site. To compare the abilities of seawater and sedimentary microbial communities to initiate organic matter degradation, we measured the extracellular enzymatic hydrolysis rates of 10 substrates (polysaccharides and algal extracts) in surface seawater and bottom water as well as in surface and anoxic sediments of an Arctic fjord. Patterns of enzyme activities differed between seawater and sediments, not just quantitatively, in accordance with higher cell numbers in sediments, but also in their more diversified enzyme spectrum. Sedimentary microbial communities hydrolyzed all of the fluorescently labeled polysaccharide and algal extracts, in most cases at higher rates in subsurface than surface sediments. In seawater, in contrast, only 5 of the 7 polysaccharides and 2 of the 3 algal extracts were hydrolyzed, and hydrolysis rates in surface and deepwater were virtually identical. To compare bacterial communities, 16S rRNA gene clone libraries were constructed from the same seawater and sediment samples; they diverged strongly in composition. Thus, the broader enzymatic capabilities of the sedimentary microbial communities may result from the compositional differences between seawater and sedimentary microbial communities, rather than from gene expression differences among compositionally similar communities. The greater number of phylum- and subphylum-level lineages and operational taxonomic units in sediments than in seawater samples may reflect the necessity of a wider range of enzymatic capabilities and strategies to access organic matter that has already been degraded during passage through the water column. When transformations of marine organic matter are considered, differences in community composition and their different abilities to access organic matter should be taken into account.

The role of fungi in processing marine organic matter in the upwelling ecosystem off Chile

In a study that spanned from March 2007 through November 2009, we report high fungal biomass and over 90% of extracellular enzymatic activity occurring in the size classes dominated by fungi during periods of high autotrophic biomass in surface waters of the upwelling ecosystem off central-southern Chile (36°30.80′S–73°07.70′W). Fungal biomass in the water column was determined by the abundance of hyphae and was positively correlated with the concentration of the fungal biomarker 18:2ω6. High fungal biomass during active upwelling periods was comparable to that of prokaryotes (bacteria plus archaea) and was associated with an increase in phytoplankton biomass and in extracellular enzymatic hydrolysis in waters from the depth of maximum fluorescence. We show fungi as a new microbial component in the coastal upwelling ecosystem of the Humboldt Current System off central Chile. Our results suggest that the temporal pattern in fungal biomass in the water column during a year cycle is a reflection of their capacity to hydrolyze organic polymers and, in consequence, fungal biomass and activity respond to a seasonal cycle of upwelling in this ecosystem.

Thermophilic anaerobes in Arctic marine sediments induced to mineralize complex organic matter at high temperature

Marine sediments harbour diverse populations of dormant thermophilic bacterial spores that become active in sediment incubation experiments at much higher than in situ temperature. This response was investigated in the presence of natural complex organic matter in sediments of two Arctic fjords, as well as with the addition of freeze-dried Spirulina or individual high-molecular-weight polysaccharides. During 50 degrees C incubation experiments, Arctic thermophiles catalysed extensive mineralization of the organic matter via extracellular enzymatic hydrolysis, fermentation and sulfate reduction. This high temperature-induced food chain mirrors sediment microbial processes occurring at cold in situ temperatures (near 0 degrees C), yet it is catalysed by a completely different set of microorganisms. Using sulfate reduction rates (SRR) as a proxy for organic matter mineralization showed that differences in organic matter reactivity determined the extent of the thermophilic response. Fjord sediments with higher in situ SRR also supported higher SRR at 50 degrees C. Amendment with Spirulina significantly increased volatile fatty acids production and SRR relative to unamended sediment in 50 degrees C incubations. Spirulina amendment also revealed temporally distinct sulfate reduction phases, consistent with 16S rRNA clone library detection of multiple thermophilic Desulfotomaculum spp. enriched at 50 degrees C. Incubations with four different fluorescently labelled polysaccharides at 4 degrees C and 50 degrees C showed that the thermophilic population in Arctic sediments produce a different suite of polymer-hydrolysing enzymes than those used in situ by the cold-adapted microbial community. Over time, dormant marine microorganisms like these are buried in marine sediments and might eventually encounter warmer conditions that favour their activation. Distinct enzymatic capacities for organic polymer degradation could allow specific heterotrophic populations like these to play a role in sustaining microbial metabolism in the deep, warm, marine biosphere.

Temperature induced decoupling of enzymatic hydrolysis and carbon remineralization in long-term incubations of Arctic and temperate sediments

Extracellular enzymatic hydrolysis of high-molecular weight organic matter is the initial step in sedimentary organic carbon degradation and is often regarded as the rate-limiting step. Temperature effects on enzyme activities may therefore exert an indirect control on carbon mineralization. We explored the temperature sensitivity of enzymatic hydrolysis and its connection to subsequent steps in anoxic organic carbon degradation in long-term incubations of sediments from the Arctic and the North Sea. These sediments were incubated under anaerobic conditions for 24 months at temperatures of 0, 10, and 20 °C. The short-term temperature response of the active microbial community was tested in temperature gradient block incubations. The temperature optimum of extracellular enzymatic hydrolysis, as measured with a polysaccharide (chondroitin sulfate), differed between Arctic and temperate habitats by about 8–13 °C in fresh sediments and in sediments incubated for 24 months. In both Arctic and temperate sediments, the temperature response of chondroitin sulfate hydrolysis was initially similar to that of sulfate reduction. After 24 months, however, hydrolysis outpaced sulfate reduction rates, as demonstrated by increased concentrations of dissolved organic carbon (DOC) and total dissolved carbohydrates. This effect was stronger at higher incubation temperatures, particularly in the Arctic sediments. In all experiments, concentrations of volatile fatty acids (VFA) were low, indicating tight coupling between VFA production and consumption. Together, these data indicate that long-term incubation at elevated temperatures led to increased decoupling of hydrolytic DOC production relative to fermentation. Temperature increases in marine sedimentary environments may thus significantly affect the downstream carbon mineralization and lead to the increased formation of refractory DOC.

Organic Carbon Degradation in Arctic Marine Sediments, Svalbard: A Comparison of Initial and Terminal Steps

Degradation of marine organic matter under anoxic conditions involves microbial communities working in concert to remineralize complex substrates to CO 2 . In order to investigate the coupling between the initial and terminal steps of this sequence in permanently cold sediments, rates of extracellular enzymatic hydrolysis and sulfate reduction were measured in parallel cores collected from 5 fjords on the west and northwest coast of Svalbard, in the high Arctic. Inventories of total dissolved carbohydrates were also measured in order to evaluate their potential role in carbon turnover. Polysaccharide hydrolysis rates exhibited substrate-related and, to a lesser extent, depth-related differences (p < 0.0001); laminarin hydrolysis was consistently most rapid at nearly all depths and sites, and fucoidan hydrolysis was least rapid. Although there was a high degree of variability in parallel cores, sulfate reduction rates also exhibited statistically significant depth-and station-related differences. A comparison with data from previous investigations in Svalbard sediments suggests that this variability is linked to substrate availability rather than to organism distribution. Total dissolved carbohydrate concentrations were comparable to those measured in more temperate sediments, and likely comprise a considerable fraction of porewater dissolved organic carbon. A comparison of dissolved carbohydrate inventories with hydrolysis and sulfate reduction rates suggests that the turnover of carbon through the dissolved pool occurs quite rapidly, on the order of a few days to weeks. The transformation of particulate to dissolved organic matter must also be sufficiently rapid to maintain the measured rates of terminal remineralization.

Speed bumps and barricades in the carbon cycle: substrate structural effects on carbon cycling

The observation that a fraction of organic matter produced in marine systems evades the concerted efforts of microbial communities and is buried in sediments suggests that there are ‘speed bumps’ in carbon degradation pathways that impede microbially driven remineralization processes. The initial step in degradation of macromolecules, extracellular enzymatic hydrolysis, is often stated to be ‘the’ rate-limiting step in carbon remineralization. Experimental investigations described here, however, demonstrate that at least in certain cases, microbes produce extracellular enzymes on time scales of hours to tens of hours in response to substrate addition, and hydrolysis is extremely rapid. If enzymatic hydrolysis can be rapid, what factors slow or stop organic matter degradation? A lack of the correct inducer to initiate enzyme production, and/or a lack of the correct organism to produce the required enzyme, may result in a complete lack of hydrolysis in certain environments—a barricade, rather than a speed bump. Preliminary evidence supporting this hypothesis includes a comparison of polysaccharide hydrolysis in seawater and sediments, which demonstrates that the spectrum of enzymes active in seawater and sediments are fundamentally different. Furthermore, a survey of enzyme activities in surface waters from a range of locations suggests that pelagic microbial communities also differ widely in their abilities to express specific extracellular enzymes. Trans-membrane transport through porins is yet another potential location of structure-related selectivity. Our efforts to identify speed bumps and barricades are hampered by our inability to structurally characterize in sufficient detail the macromolecular structures present in marine systems. Furthermore, assessments of organic matter ‘quality’ from a chemical perspective do not necessarily accurately reflect the availability of organic carbon to microbial communities. For these communities, in fact, ‘quality’ may be a variable, which depends on the enzymatic and uptake capabilities of community members. To begin to assess substrate structure and quality from a microbial perspective, we will have to combine specific knowledge of macromolecular structures with detailed investigations of the enzymatic and transport capabilities of heterotrophic marine microbes.

Decomposition of sinking proteinaceous material during fall in the oxygen minimum zone off northern Chile

We investigated the fate of sinking proteinaceous material in the oxygen minimum zone off northern Chile by deploying sediment traps at 30 m (base of the oxygenated layer) and 300 m (bottom of the O 2-depleted layer) during a 3-day experiment. Most of photosynthetically produced protein (82%) degraded in the top 30 m; an additional 15% decayed between 30 and 300 m, within the suboxic zone; and ca. 1% reached surface sediments at 1200 m depth. Sinking protein remained diagenetically labile in the top 300-m of the water column, as indicated by degradation indices and degradation rate constants of trap material, both characteristic of fresh material. We conclude that particulate protein degradation is not affected by the occurrence of the suboxic layer between 30 and 300 m in the water column. This conclusion is consistent with a model of degradation of particulate protein controlled by extracellular enzymatic hydrolysis and not dependent on O 2 availability. Assuming that our fall results are representative for an annual cycle and the whole oxygen minimum zone, suboxic decay of sinking protein in the oxygen minimum zone could support a production of 2 Tg N 2 yr −1, consistent with independent estimates of denitrification rates in the area.

Carbon cycling in a continental margin sediment: contrasts between organic matter characteristics and remineralization rates and pathways

Although particulate organic carbon (POC) in sediments is derived from diverse sources, characteristics of bulk POC are frequently used as indicators of the ‘quality’ of organic matter potentially available to sedimentary microbial communities. In order to investigate the extent to which characteristics of POC relate to sedimentary metabolism, the rates of the initial and terminal steps of organic carbon remineralization (extracellular enzymatic hydrolysis, and sulfate, iron, and manganese reduction, respectively) were compared at three sites in Skagerrak dominated by different terminal remineralization processes. In parallel, dissolved organic carbon (DOC) inventories and bulk POC characteristics were assessed. At all three sites, bulk characteristics of POC were similar, with C/N close to 12, low sedimentary amino acid content, and moderate concentrations of total hydrolyzable carbohydrates. On average, just 12% of POC was characterizable as carbohydrates or amino acids. These characteristics are frequently considered typical of unreactive or ‘low quality’ organic matter. At all three sites, however, organic carbon remineralization (measured as CO 2 production and sediment O 2 uptake) was quite high relative to other locations with similar bulk characteristics. A comparison of DOC inventories with rates of terminal remineralization demonstrated that at the three sites, on average 27, 8, and 31% of the sedimentary DOC pool must be turned over on a daily basis in order to support terminal respiration. Extracellular enzymatic activity, calculated as potential carbon turnover, was sufficient to support these rates. At these sites, standard chemical characterization of bulk POC does not reflect the reactivity and availability of substrates to the sedimentary microbial community. Carbon remineralization is likely fueled by a small fraction of POC, not distinguishable by measurement of bulk parameters, which is rapidly cycled through the DOC pool.